leupeptins and phorbolol-myristate-acetate

Polymorphic mononuclear neutrophils (PMN) are very important cells participating in nonspecific defense of the organism. Among their well-known functions, the formation of neutrophil extracellular traps (NET) is interesting and potentially dangerous for the mechanisms of other cells. Ubiquitin-dependent proteasomal proteolysis is a very important regulator of all cellular activities, but the role of proteasomal proteolysis in NET formation has not been investigated.. We performed experiments with PMN activated to form NET with phorbol 12-myristate 13-acetate (PMA) and the application of a proteasome inhibitor. We also added activated neutrophils to primary culture of isolated rat neonatal cardiomyocytes with or without anoxia-reoxygenation modeling.. The data obtained show that proteasomes participate in NET formation and proteasome inhibitors facilitate the blocking of the NET program. The percentage of NET after PMA application was 70.8 ± 7.2 and the proteasome inhibitor decreased this amount to 4.7 ± 0.9%. In coculture with cardiomyocytes during anoxia-reoxygenation, this effect prevented cardiac cell death induced by activated PMN. The stimulation of NET formation by PMA in coculture with isolated cardiomyocytes led to an increase in the number of necrotic cardiomyocytes of up to 33.1 ± 12.9% and a corresponding decrease in living cardiomyocytes to 66.9 ± 12.9%. The number of living cardiomyocytes in coculture after incubation with both PMA and proteasome inhibitor was 76.6 ± 13.3% (p < 0.05), and the number of necrotic cardiomyocytes was 23.4 ± 13.3% (p < 0.05).. Proteasome inhibition blocks NET formation and prevents cardiomyocyte necrosis in coculture with activated neutrophils.

Topics: Animals; Apoptosis; Cell Hypoxia; Cells, Cultured; Coculture Techniques; Cysteine Proteinase Inhibitors; Extracellular Traps; Leupeptins; Myocytes, Cardiac; Neutrophil Activation; Neutrophils; Proteasome Endopeptidase Complex; Rats; Tetradecanoylphorbol Acetate

2'-5' Oligoadenylate (2-5A)-dependent RNase L is one of the key enzymes involved in the molecular mechanisms of interferon (IFN) function. Although the regulation of RNase L by 2-5A has been studied extensively, relatively little is known about how RNase L is controlled by posttranslational processes. Here, we report that phorbol-12-myristate-13-acetate (PMA) treatment of mouse L929 fibroblasts caused rapid degradation of RNase L in a dose-dependent and time-dependent manner. RNase L levels were decreased to 40% of control levels after only 5 min exposure of cells to PMA, suggesting the involvement of protein kinase C (PKC). After PMA treatment for 1 h, RNase L levels decreased to 18% of the pretreatment levels. Decay of RNase L was measured by 2-5A binding assay, ribonuclease activity, and protein levels in Western blots probed with antibody to murine RNase L. PMA treatment caused decreases in the levels of RNase L in both cytoplasm and nucleus. To explore the mechanism of RNase L degradation, we treated cells with the selective proteasome inhibitors, ALLN, MG132, and PSI, prior to PMA treatment. These inhibitors completely blocked the degradation of RNase L caused by PMA. Our results show a novel regulatory pathway for RNase L that could have an impact on its antitumor and antiviral functions.

Topics: Animals; Apoptosis; Cell Line; Cell Nucleus; Cysteine Endopeptidases; Cytoplasm; Down-Regulation; Endoribonucleases; Interferon-alpha; Leupeptins; Mice; Multienzyme Complexes; Oligopeptides; Proteasome Endopeptidase Complex; Protein Transport; Tetradecanoylphorbol Acetate

Interleukin (IL)-1 beta increases beta(2)-adrenergic receptor (beta(2)-AR) mRNA and density by protein kinase C (PKC)-dependent mechanisms in human airway epithelial cells. The present study examined the role of several nuclear transcription factors in the PKC-activated upregulation of beta(2)-AR expression. BEAS-2B cells were exposed to the PKC activator phorbol 12-myristate 13-acetate (PMA; 0.1 microM for 2-18 h). PMA had no effect on activator protein (AP)-2 or cAMP response element binding protein DNA binding activity but markedly increased nuclear factor (NF)-kappa B and AP-1 binding as assessed by electrophoretic gel mobility shift assay. PMA also increased the activity of a beta(2)-AR promoter-luciferase reporter construct in transiently transfected cells. These effects were inhibited by the PKC inhibitors Ro-31-8220 and calphostin C. Furthermore, with increasing Ro-31-8220, beta(2)-AR promoter-reporter activity correlated closely with both NF-kappa B and AP-1 activities (r > 0.89 for both). Finally, the selective NF-kappa B inhibitor MG-132 dose dependently reduced NF-kappa B binding and beta(2)-AR promoter activity but increased AP-1 binding. We conclude that PKC-induced upregulation of beta(2)-AR expression in human airway epithelial cells appears to be mediated, at least in part, by increases in NF-kappa B activity.

Topics: Cell Line; Enzyme Inhibitors; Epithelial Cells; Genes, Reporter; Humans; Indoles; Interleukin-1; Leupeptins; Naphthalenes; NF-kappa B; Promoter Regions, Genetic; Protein Kinase C; Receptors, Adrenergic, beta-2; Respiratory Mucosa; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Up-Regulation